An RND transporter in the monoterpene metabolism of Castellaniella defragrans

The betaproteobacterium Castellaniella defragrans 65Phen grows on monoterpenes at concentrations toxic to many bacteria. Tolerance mechanisms include modifications of the membrane fatty acid composition and the mineralization of monoterpenes. In this study, we characterized an efflux transporter associated to the monoterpene metabolism. The inner-membrane transporter AmeD (apolar monoterpene efflux) affiliated to the HAE3 (hydrophobe/amphiphile efflux) family within the Resistance-Nodulation-Division (RND) superfamily. RND pumps of the HAE3 family are known for transporting substrates into the periplasm. AmeD is co-expressed with the outer membrane protein AmeA and the periplasmic proteins AmeB and AmeC, suggesting an export channel into the environment similar to HAE1-type RND exporters. Proteins AmeABCD are encoded within a genetic island involved in the metabolism of acyclic and cyclic monoterpenes. The deletion of ameABCD translated into a decrease in tolerance to monoterpenes in liquid cultures. The addition of acetate as cosubstrate in limonene-containing cultures partially alleviated monoterpene toxicity in the deletion mutant. Accumulation of Nile Red in cells of C. defragrans required dissipation of the proton motive force with carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells lacking AmeABCD accumulated more Nile Red, suggesting an export function of the proteins. Our observations suggest that the tetrapartite RND transporter AmeABCD acts as an exporter during monoterpene detoxification in C. defragrans

Anaerobic Degradation of Bicyclic Monoterpenes in Castellaniella defragrans

The microbial degradation pathways of bicyclic monoterpenes contain unknown enzymes for carbon-carbon cleavages. Such enzymes may also be present in the betaproteobacterium Castellaniella defragrans, a model organism to study the anaerobic monoterpene degradation. In this study, a deletion mutant strain missing the first enzyme of the monocyclic monoterpene pathway transformed cometabolically the bicyclics sabinene, 3-carene and alpha-pinene into several monocyclic monoterpenes and traces of cyclic monoterpene alcohols. Proteomes of cells grown on bicyclic monoterpenes resembled the proteomes of cells grown on monocyclic monoterpenes. Many transposon mutants unable to grow on bicyclic monoterpenes contained inactivated genes of the monocyclic monoterpene pathway. These observations suggest that the monocyclic degradation pathway is used to metabolize bicyclic monoterpenes. The initial step in the degradation is a decyclization (ring-opening) reaction yielding monocyclic monoterpenes, which can be considered as a reverse reaction of the olefin cyclization of polyenes

Limonene dehydrogenase hydroxylates the allylic methyl group of cyclic monoterpenes in the anaerobic terpene degradation by Castellaniella defragrans

The enzymatic functionalization of hydrocarbons is a central step in the global carbon cycle initiating the mineralization of methane, isoprenes, and monoterpenes, the most abundant biologically produced hydrocarbons. Also, terpene-modifying enzymes have found many applications in the energy-economic biotechnological production of fine chemicals. Here, we describe a limonene dehydrogenase that was purified from the facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen grown on monoterpenes under denitrifying conditions in the absence of molecular oxygen. The purified limonene:ferrocenium oxidoreductase activity hydroxylated the methyl group of limonene (1-methyl-4-(1-methylethenyl)-cyclohex-1-ene) yielding perillyl alcohol ([4-(prop-1-en-2-yl)cyclohex-1-en-1-yl]methanol). The enzyme had a DTT:perillyl alcohol oxidoreductase activity yielding limonene. Mass spectrometry and molecular size determinations revealed a heterodimeric enzyme comprising CtmA and CtmB. Recently, the two proteins had been identified by transposon mutagenesis and proteomics as part of the cyclic terpene metabolism (ctm) in C. defragrans and are annotated as FAD-dependent oxidoreductases of the protein domain family phytoene dehydrogenases and related proteins (COG1233). CtmAB is the first heterodimeric enzyme in this protein superfamily. Flavins in the purified CtmAB are oxidized by ferrocenium and are reduced by limonene. Heterologous expression of CtmA, CtmB, and CtmAB in Escherichia coli demonstrated that limonene dehydrogenase activity required both subunits, each carrying a flavin cofactor. Native CtmAB oxidized a wide range of monocyclic monoterpenes containing the allylic methyl group motif (1-methyl-cyclohex-1-ene). In conclusion, we have identified CtmAB as a hydroxylating limonene dehydrogenase and the first heteromer in a family of FAD-dependent dehydrogenases acting on allylic methylene or methyl CH-bonds. We suggest placing in Enzyme Nomenclature as new entry EC 1.17.99.8

The anaerobic linalool metabolism in Thauera linaloolentis 47 Lol

Background: The betaproteobacterium Thauera linaloolentis 47Lol(T) was isolated on the tertiary monoterpene alcohol (R,S)-linalool as sole carbon and energy source under denitrifying conditions. Growth experiments indicated the formation of geraniol and geranial. Thus, a 3,1-hydroxyl-Delta(1)-Delta(2)-mutase (linalool isomerase) activity may initiate the degradation, followed by enzymes of the acyclic terpene utilization (Atu) and leucine/isovalerate utilization (Liu) pathways that were extensively studied in Pseudomonas spp. growing on citronellol or geraniol. Results: A transposon mutagenesis yielded 39 transconjugants that could not grow anaerobically on linalool and nitrate in liquid medium. The deficiencies were apparently based on gene functions required to overcome the toxicity of linalool, but not due to inactivation of genes in the degradation pathway. Growing cultures formed geraniol and geranial transiently, but also geranic acid. Analysis of expressed proteins detected several enzymes of the Atu and Liu pathways. The draft genome of T. linaloolentis 47Lol(T) had atu and liu genes with homology to those of Pseudomonas spp.. Conclusion: The in comparison to monoterpenes larger toxicity of monoterpene alcohols is defeated by several modifications of the cellular structure and metabolism in Thauera linaloolentis 47Lol(T). The acyclic terpene utilization pathway is used in T. linaloolentis 47Lol(T) during growth on (R,S)-linalool and nitrate under anoxic conditions. This is the first experimental verification of an active Atu pathway outside of the genus Pseudomonas